Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 2016 The Role of c-Abl Kinase in HCC Development Lennox Chitsike Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Part of the Molecular Biology Commons Recommended Citation Chitsike, Lennox, "The Role of c-Abl Kinase in HCC Development" (2016). Master's Theses. 3259. https://ecommons.luc.edu/luc_theses/3259 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2016 Lennox Chitsike LOYOLA UNIVERSITY CHICAGO THE ROLE OF C-ABL KINASE IN HCC DEVELOPMENT A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF MASTER OF SCIENCE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY BY LENNOX CHITSIKE CHICAGO, ILLINOIS AUGUST, 2016 I dedicate this thesis to my mother ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my Ph.D advisor and mentor, Dr. Qiu for his tutelage, guidance, and support for the past year I have been in his lab. I could not have achieved half without the assistance. Secondly, I would like to express my sincere gratitude to the members of my thesis committee: Dr. Mitchell Denning and Dr. Zeleznik-Le for their insightful advice and suggestions that gave my study better perspective and new directions. Last but not least, I would like also to express my sincere gratitude to my lab members Dr. Na Shang, Maribel Arteaga and Fang Wang for helping with experimental procedures and acclimatizing to the culture of the lab. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF FIGURES vii ABSTRACT viii CHAPTER 1: INTRODUCTION 1.1 HCC incidence, mortality and clinical management 1 1.1.2 HCC etiology and progression 3 1.1.3 Sorafenib as systemic therapy drug for HCC 5 1.2 c-Abl kinase 6 1.2.1 c-Abl structure and function 6 1.2.2 c-Abl Regulation 9 1.2.3 c-Abl activation in cancer 10 1.3 Axl 12 1.3.1 Axl and its role in cancer 12 1.3.2Axl activation and functional cascades 15 1.4 Project Rationale and Aims 15 1.4.1Rationale and Research Strategy 15 1.4.2 Hypothesis 18 1:4.3 AIMS 18 CHAPTER 2: INHIBITING C-ABL EXPRESSION IN HUH7 CELL LINE ATTENUATES CELL GROWTH, INVASION, MIGRATION AND THESE PROCESSES SEEM TO BE MEDIATED THROUGH AXL 19 2.1 INTRODUCTION 19 2.1.1Materials and Methods 20 2.3 Results 24 2.3.1 c-Abl is necessary for cell growth and proliferation in vitro 24 2.3.2 c-Abl promotes cell migration in HCC 25 2.3.3 c-Abl promotes cell invasion in HCC 25 2.3.4 c-Abl promotes cell growth and proliferation in xenograft in vivo mouse model 27 2.3.5 c-Abl function is partially mediated by Axl 28 2.4 Discussion and Conclusions 29 v CHAPTER 3: Inhibiting c-Abl activation attenuates tumor cell growth and proliferation in vivo 30 3.1 Introduction 30 3.2 Materials and Methods 31 3.3 Results 33 3.3.1 c-Abl activity is necessary for cell growth and proliferation in vivo 33 3.4 Discussion and Conclusions 34 CHAPTER 4: c-Abl knockdown has synergistic effects with Sorafenib in inhibiting HCC growth in vitro and in vivo 36 4.1 Introduction 36 4.2 Materials and Methods 37 4.3 Results 37 4.31 c-Abl knockdown enhances Sorafenib growth inhibition and apoptosis induction in vitro 37 4.3.2 c-Abl knockdown enhances Sorafenib-mediated HCC cell growth inhibition in vivo 39 4.3.3 Sensitization of c-Abl KD cells to Sorafenib treatment occur through a c-Abl-Axl axis 42 4.4 Discussion and Conclusions 44 BIBLIOGRAPHY 47 VITA 51 vi LIST OF FIGURES Figure 1: EASL staging and clinical practice guidelines management of hepatocellular-carcinoma 2 Figure 2: Schematic of environmental, genetic etiological factors and molecular changes that occur during HCC development 4 Figure 3: Modular structure of c-Abl 6 Figure 4: Chemical structures of Imatinib, Nilotinib, Dasatinib and Sorafenib 8 Figure 5: Schematic representation of downstream events upon binding of Gas6 to Axl in different cells 14 Figure 6: c-Abl overexpression and hyperactivation is associated with poor clinical prognosis 17 Figure 7: c-Abl and Axl shRNA sequences 20 Figure 8: c-Abl promotes cell growth and proliferation in HCC in vitro 24 Figure 9: c-Abl promotes cell invasion and migration in HCC in vitro 26 Figure 10: c-Abl promotes cell growth and proliferation in HCC in xenograft mouse model 27 Figure 11: Axl is the major target and mediator of c-Abl in HCC huh7 cell line 29 Figure 12: Met/Cat HCC liver cancer mouse model scheme 32 Figure 13: Activated c-Abl promotes cell growth and proliferation in HCC in vivo 34 Figure 14: c-Abl inhibition sensitizes HCC cells to Sorafenib treatment and Sorafenib-induced apoptosis in vitro 39 Figure 15: c-Abl inhibition sensitizes HCC cells to Sorafenib treatment in vivo 41 Figure 16: Sensitization of c-Abl KD cells to Sorafenib treatment occur through c-Abl-Axl axis 43 vii Abstract of Thesis Hepatocellular Carcinoma (HCC) is the second most lethal cancer after pancreatic cancer, killing almost a million people worldwide every year. Unresectable HCC tumors (intermediate to advanced stage) carry a poor prognosis and very few treatment options are available. The dismal prognosis is mainly due to limited therapy options with Sorafenib as the only approved FDA-approved drug to treat HCC. Molecularly targeted therapy against important HCC oncogenic drivers has been proposed as solution to the problem. Here, we report a novel role of c-Abl in HCC development. We provide evidence of c-Abl activation in human HCC samples compared to normal human liver samples. Using complimentary genetic and pharmacological tools, we show that c-Abl plays a vital role in promoting cell growth, proliferation, migration and invasion in vitro and tumor growth in vivo. We have identified Axl as a possible effector in these processes mediated by c-Abl. Our findings also show that c-Abl inhibition has synergistic effects with Sorafenib treatment and that this synergism occurs because Sorafenib induces activation of both c-Abl and Axl and their downstream targets. Taken together, these data we have thus far provide evidence for c-Abl as a potential critical oncogenic driver that maybe a viable target for HCC therapy, either alone or in combination with Sorafenib. Key words: c-Abl, proliferation, migration, invasion, chemosensitization viii Chapter 1: INTRODUCTION 1.1 HCC incidence, mortality and clinical management Hepatocellular carcinoma (HCC) is the most aggressive form of liver cancer, comprising 83% of all liver cancer cases. Globally, it is the 5th commonest malignancy in the world and is second only to lung cancer in total cancer-related deaths annually. Even though it used to be confined mainly to developing countries, which made it less of a priority in the scientific community, HCC incidence has been on the rise in Europe and the US in the past decades, with the rates more than doubling in the US. What makes the case for HCC even more pressing is the fact that it is the second most lethal cancers (8.9% 5 year survival in the US) after renal cell carcinoma (4.4% 5 year survival). 1, 2 Surgical resection, liver transplantation and ablation are the only curative therapies available but they are only available for 30% of HCC patients with very early and early stage HCC (Figure1). 80% of HCC cases are presented late because early stage liver is asymptomatic and no reliable biomarkers are available to identify patients with incipient HCC. For those already with the disease imaging and AFP, a biomarker in about 50-70% of patients, are usually used for diagnosis. Unfortunately, patients who undergo tumor resection tend to be also associated with recurrence (50% in 2 years) and for some patients eligible for living donor liver transplants, the wait time can be long and die before a matching donor is found. For HCC patients diagnosed with intermediate stage HCC and functional livers, transcatheter arterial chemoembolization (TACE) is their only option. TACE involves injection of cytotoxic drugs into the 1 2 arteries that feed tumor vasculature. In cases where intermediate HCC patients have dysfunctional liver or HCC is in advanced stage, Sorafenib becomes the only option (Figure 1).3-8 Figure 1. EASL staging and clinical practice guidelines management of hepatocellular-carcinoma. Sorafenib (circled in red) is the only FDA-approved drug for HCC. (Adapted from Forner. A et al).3 3 1.1.2 HCC etiology and progression HCC is a very complex disease whose etiologies originate from chronic HBV and HCV infections, chronic alcohol consumption, aflatoxin and various other causes of liver injury that result in liver cirrhosis and excessive accumulation of collagen. HCC then progresses due to changes in a number of genetic and epigenetic profiles that affect key proto-oncogenic pathways. Genomic integration of HBV can lead to HCC in a number of ways, including microdeletion of genes that are related to cancer or inactivation of tumor suppressor genes. Alternatively, the Hbx viral protein can alter the transcriptional profiles of genes related to cell growth. HCV can also induce HCC in a similar fashion by inactivating tumor suppressor genes, have its core proteins interact with effectors of the cell’s standard proliferative pathways or causing ER stress.